1. Quantitative Microscopy Morphogenesis using Nikon NIS, ImagePro Stella Breslin Biosc3 Spring 2010

2. Nikon Manual Count Run the [View > Analysis Controls > Annotations and Measurements] command. The manual measurement control window appears. The Auto Detect filled tool. Detect hollows using threshold techniques. Click to the image to place the probe, the detected area is drawn. You can adjust the thresholding range by using the mouse wheel or by pressing the +, - keys. [Ctrl+B]

3. Nikon Manual Count

4. Nikon Automated Measurement Optical system calibration. Optical system calibration. Image acquisition. Image acquisition. Defining threshold, creating the binary layer. Defining threshold, creating the binary layer. Performing the automated measurement. Performing the automated measurement. Results presentation. Results presentation.

5. Binary Layer Binary layer] - is typically used for shape and size measurements (area, perimeter, surface fraction).

6. Binary Layer - Manipulation

7. Automated Measurements [View > Analysis Controls > Automated Measurement]

8. Object Count

9. Nikon Threshold By Reference Points – 1 Point, 3 Points (circle), 6 Points Reset, Undo, Redo,

10. Restrictions Right click to the restrictions field to select one or more of the available measurement features. Right click to the restrictions field to select one or more of the available measurement features. Select the restriction feature you would like to define. Select the restriction feature you would like to define.

11. Move sliders below the histogram to set the limits. The limit values are indicated next to the feature name, and can be modified by double clicking the indicated value. Decide whether the defined interval will be excluded or included - this is done by setting the [Inside/Outside] option next to the feature name. The nearby check box indicates whether the restriction is currently being applied or not. If applied, the histogram below is color, otherwise it is gray.

12. Separating Objects

13. Measurement Menu Measurement Menu > Perform Measurement Object Features … Options

14. Nikon1 Object Count – No Calibration Needed Since nd2 File Sort By Area to determine Restriction By Area Size, etc

15. Measurement Controls Results of all automated measurements are presented and can be exported from the [View > Analysis Controls >Automated Measurement Results] control window. Results of all automated measurements are presented and can be exported from the [View > Analysis Controls >Automated Measurement Results] control window.

16. Results – Can be Exported to Excel or Text [ Total Area] is the area of the whole image in px2 [ Area Fraction] is the ratio between [Total Area] area of the thresholded objects. [ Number of objects] -after restrictions. [ Table of results] - measurement results [ #] - number of selected objects

17. Counting In Image Pro

18. The basic steps in counting 1. Set the range of intensities (monochrome images) or the colors (color images) that identify the object you want to count using the tools in the Intensity Range Selection group box.

19. Manual Intensities – Select Ranges Use this dialog box to segment out the objects that are to be counted and/or measured. Use this dialog box to segment out the objects that are to be counted and/or measured.

20. Manual Intensities – Select Ranges

21. 2. Select the kinds of measurement you want to record in the Count/Size : Measure menu’s Select Measurements command.

22. Count / Size Options Set Outline Style – Fill, Outline, etc Set Outline Style – Fill, Outline, etc Label Style Label Style Label Color Label Color Fill Holes Fill Holes Clean Border options Clean Border options

23. Object Options:. Clean Border: Select an option from the drop-down list to exclude from the count any objects that intersect the designated edges of your image or AOI. Pre-filter: Select this option to include in the Total Count display, only those objects that are within the specified range. When Pre-filter is not checked, the Total Count value represents all objects within the AOI or image, not just those meeting the range criteria. Fill Holes: Select this option to exclude from the count any objects that are embedded within other objects. Convex hull: Select this option to create a smooth area aroundthe perimeter of irregularly shaped objects

24. ImagePro Count 3. Count and measure the objects using the Count button

25. ImagePro Count Measure Objects – record measurements with Counts Apply Ranges Clear Counts

26. Edit Menu –Split Objects Split Manually – line, freeform Auto-Split Watershed Split Limited Watershed Split Draw /Merge Command Draw AOI Convert AOI to Object

27. Edit Menu Toggle Objects On/Off: Use this command to show or hide objectsin your image. When you left-click the mouse cursor on an object, its overlay is hidden, so that object will not be taken into account in any measurements or statistics. Left-click the mouse cursor again to display the object. Delete Hidden Objects: Use this command to remove objects that you have hidden, or that are out of the selected range. Click Yes to remove the objects. The remaining objects in the image will be renumbered.

28. Apply to Multichannel Image using File>Save Outlines File> Load Outlines

29. Measure Data Sort Locate the Object – highlights selected object in image

30. Measure Data - Sort

31. Count / View Menu Locate the Object – highlights selected object in image

32. Exported Table File Menu Data to File Append Data to file Data to Clipboard Table to Clipboard Export Data